以保罗样激酶1为靶点的抗肿瘤药物的筛选及活性评价

doi:10.3969/j.issn.1006-7795.2014.03.015

首都医科大学学报 ›› 2014, Vol. 35 ›› Issue (3): 337-343.doi: 10.3969/j.issn.1006-7795.2014.03.015

以保罗样激酶1为靶点的抗肿瘤药物的筛选及活性评价

张晶, 刘伟, 陈云雨, 司书毅

中国医学科学院医药生物技术研究所国家新药(微生物)筛选实验室, 北京 100050

收稿日期:2014-02-20 出版日期:2014-06-21 发布日期:2014-06-14
通讯作者: 司书毅，E-mail：sisyimb@hotmail.com E-mail:sisyimb@hotmail.com
基金资助:
国家自然科学基金青年科学基金（81001387）。

Screening and activity evaluation for potential inhibitors of human PLK1

Zhang Jing, Liu Wei, Chen Yunyu, Si Shuyi

Institute of Medicinal Biotechnology Academy of Medical Sciences & Peking Union Medical College, The National Laboratory for Screening New (Microbial) Drugs(LSMD), Beijing 100050, China

Received:2014-02-20 Online:2014-06-21 Published:2014-06-14
Supported by:
This study was supported by National Natural Science Funds of China for the Youth(81001387).

摘要/Abstract

摘要：

目的利用酵母模型进行保罗样激酶1（Polo-like kinase 1，PLK1）抑制剂的高通量筛选，并初步评估活性化合物的体外抗肿瘤效果。方法采用噻唑蓝比色法（MTT法）检测初筛阳性化合物对不同细胞系增生的影响，酶联免疫吸附测定法（enzyme-linked immunosorbent assay，ELISA）分析化合物对体外纯化的PLK1激酶的作用效果，流式细胞术（flow cytometry，FCM）检测细胞周期分布和凋亡率，免疫印迹（Western blotting）检测化合物对周期蛋白B1（cyclin B1）和PLK1的作用。结果利用酵母模型初筛得到3个阳性化合物，MTT法检测显示1号和3号化合物能抑制多种肿瘤细胞的增生而对正常细胞的毒性很小，ELISA结果显示2号和3号化合物对体外纯化的PLK1几乎无抑制作用，FCM检测显示1号化合物处理组G2/M期细胞比例高于对照组，Western blotting表明1号化合物不影响PLK1的蛋白表达，能导致cyclin B1的表达增加。结论 1号化合物通过抑制PLK1激酶的活性发挥抗肿瘤作用而不影响PLK1的蛋白表达，有望成为靶向PLK1的抗肿瘤先导化合物。

关键词: 保罗样激酶1抑制剂, 抗肿瘤, 细胞周期, 凋亡, 蛋白免疫印迹

Abstract:

Objective To find potential small-molecular PLK1 inhibitors through high throughput screening using budding yeast assay and examine the in vitro anti-tumor effects of these compounds. Methods The influences of the positive compounds on cells' proliferation were measured by MTT assays. ELISA assay was employed to explore their inhibition activities on purified recombinant human PLK1. Flow cytometry(FCM) was conducted to determine their effects on cell cycle and apoptosis in Hela cells. The levels of Cyclin B1 and PLK1 were detected by Western blotting. Results We got 3 hits from preliminary screening by yeast assay. In vitro antitumor results revealed that compounds 1 and 3 showed the enhanced inhibition on the cell proliferation of human cancer cell lines with concentration increasing, while the activity of compound 2 was very weak. Compound 1 had lower nanomolar activity against the PLK1 enzyme without influencing its protein expression compared with compound 2 and 3. FCM showed that cells treated with compound 1 were arrested in G2/M phase. Western blotting showed an increase in expression of Cyclin B1 in Hela cells after treated with compound 1. Conclusion The inhibition effect of compound 1 on PLK1 was associated with kinase activity without influencing the PLK1 protein expression. Compound 1 is expected to become one of the anti-cancer drug candidates targeted at PLK1.

Key words: Polo-like kinase 1 inhibitor, anti-cancer, cell cycle, apoptosis, Western blotting

中图分类号:

张晶, 刘伟, 陈云雨, 司书毅. 以保罗样激酶1为靶点的抗肿瘤药物的筛选及活性评价[J]. 首都医科大学学报, 2014, 35(3): 337-343.

Zhang Jing, Liu Wei, Chen Yunyu, Si Shuyi. Screening and activity evaluation for potential inhibitors of human PLK1[J]. Journal of Capital Medical University, 2014, 35(3): 337-343.

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以保罗样激酶1为靶点的抗肿瘤药物的筛选及活性评价

Screening and activity evaluation for potential inhibitors of human PLK1

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